采用AlphaScreen® 试剂盒(PerkinElmer)进行cAMP累积试验

Cassandra Koole; Denise Wootten; Patrick Sexton

doi:10.21769/BioProtoc.805

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Peer-reviewed

cAMP Accumulation Assays Using the AlphaScreen^® Kit (PerkinElmer)

采用AlphaScreen® 试剂盒(PerkinElmer)进行cAMP累积试验

CK Cassandra Koole email

DW Denise Wootten

PS Patrick Sexton

发布: 2013年07月05日第3卷第13期 DOI: 10.21769/BioProtoc.805 浏览次数: 12788

评审: Cheng Zhang

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参见作者原研究论文

The authors used this protocol in:

Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Nov 2012

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Abstract

Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated G_s protein-coupled receptors. Conversely, formation of cAMP is inhibited by G_i protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing G_s-coupled receptors.

Materials and Reagents

Sterile 96-well clear flat bottom plates (BD Biosciences, Falcon^®, catalog number: 353072 )
Phenol Red Free Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco^®, catalog number: 21063-029 )
Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 )
Tween-20 (Sigma-Aldrich, catalog number: P2287 )
HEPES (Life Technologies, Gibco^®, catalog number: 11344-041 )
100% Ethanol
Milli-Q H₂O
Alphascreen^® cAMP Assay Kit (PerkinElmer, catalog number: 6760625 )
White OptiPlate-384 well microplate (PerkinElmer, catalog number: 6007290 )
TopSeal (PerkinElmer, catalog number: 6005250 )
Stimulation buffer (see Recipes)
Lysis buffer (see Recipes)
Acceptor buffer (see Recipes)
Donor buffer (see Recipes)

Equipment

Fusion-α plate reader or Envision plate reader with appropriate alphascreen modules (PerkinElmer)
Humidified Incubator
Multichannel Pipettes
Micropipettes
Benchtop centrifuge
Oven
Orbital shake

Procedure

English

中文翻译

文章信息

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如何引用

Koole, C., Wootten, D. and Sexton, P. M. (2013). cAMP Accumulation Assays Using the AlphaScreen^® Kit (PerkinElmer) . Bio-protocol 3(13): e805. DOI: 10.21769/BioProtoc.805.